1. Field of the Invention
The invention relates in general to the monitoring of chemical reactions during automated synthesis and more specifically in a preferred embodiment to an online system and method for monitoring, for example, fluorenylmethyloxycarbonyl (Fmoc) deprotections during peptide synthesis.
2. Description of the Related Art
It is known in the art to synthesize biological products through automated synthesis. For example, automatic apparatus for solid phase peptide synthesis (SPPS) have become very popular for the production of naturally occurring and artificial peptides and proteins. The general principle of SPPS is one of repeated cycles of coupling and deprotection. In essence, the free N-terminal amine of a peptide attached to a solid-phase support is coupled to the carboxyl end of a single N-terminal protected amino acid. This newly coupled amino acid is then deprotected, revealing a new N-terminal amine to which a further protected amino acid may be attached.
The 9-fluorenylmethyloxycarbonyl (Fmoc) group is commonly used as a protecting group for primary and secondary amines. The Fmoc group can be incorporated by reacting the amine with reagents of the general structure Fmoc-X, such as 9-fluorenylmethyl chloroformate (Fmoc-Cl) and 9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu). Once attached, the Fmoc protecting group is stable to acid, but labile to bases such as piperidine (see below).
These characteristics are particularly useful in solid phase peptide synthesis because the removal of the Fmoc group with piperidine does not interfere with the acid labile linker attaching the peptide to the solid phase.References:                1) Chinchilla R, Dodsworth D J, Najera C, Soriano J M. A new polymer supported reagent for the Fmoc-protection of amino acids. Tet. Lett. 2001; 42: 7579-7581.        2) Paquet A. Introduction of 9-fluorenylmethyloxycarbonyl, trichloroethoxycarbonyl, and benzyloxycarbonyl amine protecting groups into O-unprotected hydroxyamino acids using succinimidyl carbonates. Can. J. Chem. 1982; 60: 976-980.        
Given that the ultimate yield of a synthesized product depends on the yield of each step of the synthesis process, the coupling of amino acids during SPPS must be highly optimized. Because the extent of deprotection is a crucial parameter in SPPS, it often must be repeated until “complete,” i.e., as much deprotection has occurred as is likely to such that further repetition is considered wasteful. Thus, various ways of monitoring the extent of the completion of the deprotection reaction have been developed.
For example, Fmoc deprotection has been monitored via conductivity assays. However, it has been found that sensitivity to conductive impurities, among other reasons, can lead to unnecessary repetition of the deprotection reaction and the resulting excess of time, reagent consumption, and lower yield.
A more sensitive approach to monitoring deprotection involves the use of ultraviolet (UV) light at 365 nm to measure the adsorption of the dibenzofulvene-piperidine adduct formed during the deprotection reaction. Unfortunately, this method also has significant drawbacks relating to artificial readings caused by undesired adsorption by other reagents such as triazole-based coupling reagents. Subsequently, the use of UV light at 301 nm to measure the adsorption of deprotection reagents and/or adducts has been found to be more advantageous.
Known apparatus and methods of UV monitoring at 301 nm involve the use of a flow cell, UV source and detector that is external to the synthesizer, and, thus, rely on moving liquid reagents from the reactor to the detector's flow cell at the conclusion of a deprotection reaction. This requires extra chemical rinsing between UV measurements to clear lines and remove bubbles. In addition, flow rates through the cell can affect the measurement accuracy and wait periods must be inserted prior to and after a reading for the values to stabilize. The extra use of reagents and extra time required make the method uneconomical at a scale greater than 1.0 mmol. Moreover, because the UV measurements are performed offline (upon conclusion of the deprotection reaction cycle), adjustments cannot be made in real time to the length of deprotection reactions, resulting in longer than necessary reaction times.
Thus, there is a need for on-line monitoring systems and methods to more efficiently produce synthesized products of high quality and yield.